Parent Proposal: This program is devoted to solving central questions in metallobiochemistry through the use of multinuclear, multifrequency (9, 35, 95 GHz) CW and pulsed electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR), and electron spin-echo envelope modulation (ESEEM) spectroscopies to characterize transition-metal centers that are vital to human health and disease. With these techniques we are able to completely characterize the active-site environment of a biological metal ion, and do so for key catalytic intermediates trapped by rapid freeze- quench and cryoreduction methods. When combined with isotopic labeling enabled by methods of modern molecular biology, these techniques permit the use of ENDOR/ESEEM to structurally characterize every stage of an enzyme's catalytic cycle. The current grant period further has shown that novel kinetic protocols can augment such structural information about intermediates with fundamental information about their reactivity. Together, these techniques provide the means to determine in precise detail how metal centers function in biology. The specific aims of this program have expanded to include: formation of bioavailable nitrogen through enzymatic reduction of N2; anabolic and catabolic activation of O2 by heme and nonheme Fe enzymes; physiological protection against oxidative stress and contrasting toxicity, afforded by 'inorganic' Mn2+; enzymatic control of radical reactions in the 'Radical SAM' (S-adenosyl methionine) enzyme superfamily; development of new ENDOR methodologies and new applications in biology. Supplement: The foundation of our effort is a 35 GHz pulsed ENDOR/ESEEM spectrometer, and the heart of this instrument is its microwave bridge. This bridge defined the state-of-the-art when it was constructed in the mid-1990s. However, time has eroded its performance in general, while key components are failing and cannot be replaced. Thus a replacement bridge is urgently needed. This replacement will further offer enhanced capabilities. With advances in microwave electronics, many of the components of the current bridge, even when new, did not match those available today. In addition, the new bridge will incorporate two microwave arms, not one as in the current bridge, enabling us to perform HYSCORE (and DEER) experiments, which were uncommon for bioinorganic systems when the current bridge was designed, but have since become standard. PUBLIC HEALTH RELEVANCE: Parent Proposal: Metal centers play a central role in human health and disease, and our program is devoted to the development and application of advanced paramagnetic resonance measurement techniques as a uniquely incisive means of revealing how biologically important metal centers function. The systems to be studied play many essential roles, including: protection from oxidative stress and reactive-oxygen species; formation of the key signaling and immune-response molecule, nitric oxide; xenobiotic and drug metabolism; antibiotic formation and resistance; regulation of diabetes-related metabolic pathways; enzymatic control of metabolically vital free-radical reactions; enzymatic formation of the bio-available nitrogen on which two-thirds of the world's population depend. Supplement: The foundation of this effort is a 35 GHz pulsed ENDOR/ESEEM spectrometer, the heart of this instrument is its microwave bridge, which is failing. his supplement requests a replacement for the bridge.